Detailed knowledge of the structures is a vital component of our understanding of the molecular basis of life. From a practical point of view, the atomic-scale structure of the protein can potentially greatly facilitate the design of effective pharmaceuticals. Modern nuclear magnetic resonance (NMR) spectroscopy continues to be a central technique in the characterization of the structure and dynamics of proteins, nucleic acids and their complexes. Ongoing advances in experimental techniques continues to push the size limits accessible by NMR and clever sample preparation methods has opened the door for the study of otherwise recalcitrant proteins such as integral membrane proteins. However, progress continues to be largely incremental, and it is clear that a radical shift in approach will likely be necessary to fully implement a knowledge-based approach to fundamental problems in human health and disease. The reverse micelle technology was originally devised to address the slow tumbling problem presented by large soluble proteins to solution NMR methods. From that initial conception it has been shown to be useful for studying a wide array of traditionally intractable proteins such as integral and anchored membrane proteins, aggregation prone proteins, and marginally stable proteins. The basic idea is to take the protein of interest and encapsulate it within the protective aqueous core of a reverse micelle particle and dissolve the entire assembly in a low viscosity fluid such as liquid ethane. In the low viscosity fluid, the reverse micelle particle tumbles faster than the protein dissolved in bulk water. This provides a significant improvement in the NMR relaxation properties governing the efficiency of the modern "triple resonance" experiments. By using this method protein constructs as large as 150 kDa can be studied without benefit of deuteration or the TROSY effect and thus more comprehensive structural and dynamical information can be obtained. To maximize this effect reverse micelle samples must be prepared in liquid ethane, which requires the preparation of samples under significant pressure and maintenance of the pressurized sample within an NMR sample tube. Daedalus Innovations has overcome the initial barrier to the implementation of this approach by developing hardware solutions for researchers to produce such samples in a safe and reproducible manner without the need for any previous experience with high-pressure applications. In this proposal we seek to develop an instrument that overcomes the current critical limitation to regular use, which is the seeming daunting task of finding encapsulation conditions for new proteins. Currently, the conditions for encapsulation (surfactant mixture;sample buffer;etc.) is optimized manually often in a material intensive manner. This is unacceptable for most non-academic applications and is certainly non-ideal in general. A more streamlined and less personnel and material intensive approach is needed. We propose to develop an instrument that will allow relatively automated examination of an array of encapsulation conditions and will identify optimum combinations using a variety of spectroscopic probes, and do so with minimal consumption or reagents. The instrument will build upon Daedalus Innovations'proven technology. The goal is to provide researchers having no intimate knowledge of the art of protein encapsulation to make use of this powerful technology. The proposed instrument will complete the suite of instruments offered by Daedalus Innovations that is designed to provide a turn-key solution for structural studies of macromolecules using the reverse micelle encapsulation strategy.
Biomedical research continues to expand the use of detailed atomic-scale structure in developing a detailed understanding of the molecular basis for life and for disease. Tools for the identification of means for intervention at the molecular level are of paramount importance. This proposal seeks to continue the development of a novel approach to structure determination by nuclear magnetic resonance. If successful, this technology could serve as a powerful platform for the rational design of pharmaceuticals for the treatment of an array of human diseases.